1. Field of the Invention
The present invention relates generally to programmed cell death and specifically to compositions and methods for ameliorating or preventing inflammation and symptoms associated therewith using interleukin-1xcex2-converting enzyme (ICE)/CED-3 family inhibitors.
2. Description of the Related Art
The present invention relates generally to programmed cell death and specifically to methods for expansion of hematopoietic cells, for prolonging viability of an organ for transplantation, and maintaining viability of cell lines used in bioproduction using interleukin-1xcex2-converting enzyme (ICE)/CED-3 family inhibitors.
Necrosis and apoptosis are two basic processes by which cells may die. In necrosis cell death usually is a result of cell injury. The cells generally swell and lyse, and the cell contents ultimately spill into the extracellular space. By contrast, apoptosis is a mode of cell death in which single cells are deleted in the midst of living tissues. Apoptosis accounts for most of the programmed cell death in tissue remodeling and for the cell loss that accompanies atrophy of adult tissues following withdrawal of endocrine and other growth stimuli. In addition, apoptosis is believed to be responsible for the physiologic death of cells in the course of normal tissue turnover (i.e., tissue homeostasis) (Kerr, J. F. et al., Br. J. Cancer 26:239-257, 1972; Wyllie, A. H. et al., Int. Rev. Cytol. 68:251-306, 1980).
The effector mechanisms of apoptosis are not completely understood, but ultimately, certain nuclear changes occur that appear to be caused by the activation of endogenous nucleases that cleave chromatin between nucleosomes and reduce the content of intact DNA in apoptotic cells. A number of regulators of apoptosis have been identified. Some of these are already familiar as protooncogenes and oncosuppressor genes, including c-myc, bcl-2, p53, and ras. The protooncogene products and oncosuppressor proteins are believed to control cellular susceptibility to apoptosis (Isaacs, J. T., Curr. Opin. Oncol. 6:82-89, 1994). C-myc can deterrnine whether cells continuously proliferate or enter apoptosis, depending on the availability of critical growth factors (Bisonnette, R. P. et al., In Apoptosis II: The Molecular Basis of Apoptosis in Disease. Cold Spring Harbor Laboratory Press, 1994). In cultured cells, proliferation is usually observed in the presence of c-myc and growth factors, whereas apoptosis is seen when c-myc is present but growth factors are absent. Certain other oncogenes (e.g., bcl-2) rescue cells from susceptibility to apoptosis. Specifically, members of the bcl-2 gene family can act to inhibit programmed cell death (e.g., bcl-2, bcl-xL, ced-9) or promote cell death (e.g., bax, bak, bcl-xS). Additionally, members of the ICE/CED-3 family can promote cell death (e.g., ICE, CPP32, Ich-1, CED3).
Interleukin 1 (xe2x80x9cIL-1xe2x80x9d) is a major pro-inflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. (Oppenheim, J. H. et al., Immunology Today 7:45-56, 1986). As such, it is involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response. (Mosely, B. S. et al., Proc. Nat. Acad. Sci, 84:4572-4576, 1987; Lonnemann, G. et al., Eur. I. Immunol. 19:1531-1536, 1989).
Mammalian IL-1xcex2 is synthesized as a precursor polypeptide of about 31.5 kDa (Linjuco, et al., Proc. Natl. Acad. Sci. USA 83:3972, 1986). Precursor IL-1xcex2 is unable to bind to IL-1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262:2941, 1987). Biological activity appears dependent upon proteolytic processing which results in the conversion of the precursor 31.5 kDa form to the mature 17.5 kDa form.
Proteolytic maturation of human precursor IL-1xcex2 to mature, 17 kDa IL-1xcex2 results from cleavage between Asp116 and Ala117. An endoproteinase, termed Interleukin-1xcex2 Converting Enzyme (ICE), has been identified in human monocytes that is capable of cleaving the IL-1xcex2 precursor at Asp116-Ala117, as well as at the site Asp27-Gly28, and generating mature IL-1xcex2 with the appropriate amino terminus at Ala117. The Asp at position 116 has been found to be essential for cleavage, since substitution of Ala (Kostura et al., Proc. Natl. Acad. Sci. 86:5227, 1989) or other amino acids (Howard et al., J. Immunol. 147:2964, 1991) for Asp inhibits this cleavage event.
The substrate specificity of human ICE has been defined with the use of peptides that span the cleavage site of the enzyme. Two features of peptide substrates are essential for catalytic recognition by the enzyme. First, there is a strong preference for aspartic acid adjacent to the cleavage site, in that any substitution of this residue in the IL-1xcex2 precursor and peptide substrates leads to a substantial reduction in the rate of catalysis (Kostura et al., Proc. Natl. Acad. Sci. 86:5227, 1989; Sleath et al., J. Biol. Chem. 265:14526, 1990; Howard et al., J. Immunol. 147:2964, 1991). There is an equally stringent requirement for four amino acids to the left of the cleavage site, whereas methylamine is sufficient to the right. The minimal substrate for the enzyme, AC-Tyr-Val-Ala-Asp-NHxe2x80x94CH3, is a particularly good peptide substrate with a relative Vmax/Km similar to that of the IL-1xcex2 precursor itself (Thomberry et al., Nature 356:768, 1992).
ICE is a cysteinyl proteinase by the following criteria: (1) the diazomethylketone AC-Tyr-Val-Ala-Asp-COCHN2 is a potent, competitive, irreversible inhibitor of the enzyme, (2) inactivation of the enzyme by iodoacetate is competitive with substrate, and (3) the catalytically active Cys reacts selectively with [14C] iodoacetate more than 10 times faster than do other cysteines or dithiothreitol (Thomberry et al., Nature 356:768, 1992).
ICE is related structurally and finctionally to the CED-3 protease that functions as a cell death effector in the roundworm C. elegans (Yuan et al., Cell 75:641, 1993). ICE and CED-3 form part of a larger family of proteases (the ICE/CED-3 family) that includes CPP32, ICH-1, Mch-2, ICErel II, ICErel III, Mch-3, Mch4 and Mch-5. All of these enzymes are cysteine proteases that share significant homology at their active sites. They also share the specificity for substrate cleavage at asp-x bonds. Additionally, each of the ICE/CED-3 family members is synthesized as a pro-enzyme that is then proteolytically activated to form an active enzyme.
Thus, disease states in which inhibitors of the ICE/ced-3 family of cysteine proteases may be useful as therapeutic agents include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, ischemic diseases such as the myocardial infarction, stroke and ischemic kidney disease; immune-based diseases, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain neurodegenerative diseases.
In various cell culture systems, it has been shown that inhibition of ICE/CED-3 family members can effectively inhibit apoptosis. For example, the compound acetyl-DEVD-aldehyde inhibited anti-Fas induced apoptosis in a T-lymphocyte cell line (Schlegel et al., J. Biol. Chem. 271:1841, 1996; Enari et al., Nature 380:723, 1996). Similarly, acetyl-YVAD-aldehyde and acetyl-YVAD-chloromethylketone blocked the death of motoneurons in vitro and in vivo (Milligan et al., Neuron 15:385, 1995). In addition, the ICE/CED-3 family inhibitor Boc-D-(benzyl) chloromethylketone as well as crmA prevented the cell death of mammary epithelial cells that occurs in the absence of extracellular matrix (Boudreau et al., Science 267:891, 1995).
It is known that control of apoptosis may have utility in treating disease. Specifically, inhibitors of the ICE/CED-3 family may have therapeutic effects. For example, it has been suggested that inhibition of ICE may be useful in the treatment of inflammatory disorders (Dolle et al., J. Med. Chem. 37:563, 1994; Thomberry et al., Biochemistry 33:3934, 1994). It is also known that inhibitors of ICE/CED-3 family members may have utility in treating degenerative diseases such as neurodegencrative diseases (e.g., Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, Huntington""s disease), ischemic disease of heart or central nervous system (i.e., myocardial infarction and stroke), and traumatic brain injury, as well as in alopecia, AIDS and toxin induced liver disease (Nicholson, Nature Biotechnology 14:297, 1996).
Peptide and peptidyl inhibitors of ICE have been described. However, such inhibitors have been typically characterized by undesirable pharmacologic properties, such as poor oral absorption, poor stability and rapid metabolism. (Plattner, J. J. and D. W. Norbeck, Drug Discovery Technologies, C. R. Clark and W. H. Moos, Eds. (Ellis Horwood, Chichester, England, 1990, pp. 92-126.) These undesirable properties have hampered their development into effective drugs. The methods of this invention include either the use of conformationally constrained dipeptide mimetics or a N-substituted indolyl peptide replacement. These mimetics exhibit improved properties relative to their peptidic counterparts, for example, such as improved absorption and metabolic stability resulting in enhanced bioavailability.
The present invention is directed to ameliorating inflammation and/or its symptoms, such as redness, irritation, itching, edema (swelling), burning, and etc. by inhibiting the activity of proteases of the interleukin-1xcex2-converting enzyme (ICE)/CED-3 family. The current invention provides new compositions and methods for using ICE/CED-3 inhibitors.
The present invention generally provides methods and compositions for preventing or treating inflammation. In one aspect, the present invention provides a method for preventing or treating inflammation by contacting a cell population with an inhibiting effective amount of a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family, thereby preventing or treating inflammation. In certain embodiments, said inflammation is chronic inflammation, acute inflammation, or due to an inflammatory disease. In a further embodiment, the inflammatory disease is selected from the group consisting of septic shock, septicemia, and adult respiratory distress syndrome. In other embodiments, the reagent suppresses the protease activity in an irreversible manner or a reversible manner. In another embodiment, the reagent is a compound or a pharmaceutically acceptable salt thereof. The compound is represented by formula 1:
wherein n is 1 or 2; R1 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH2)mCO2R4, wherein m=1-4, and R4 is as defined below; R2 is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH2)pCO2R5, wherein p=0-4, and R5 is as defined below; R3 is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R4 is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R5 is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH2ZR6, CH2OCO(aryl), CH2OCO(heteroaryl), or CH2OPO(R7)R8, wherein Z is an oxygen or a sulfur atom; R6 is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; R7 and R8 are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl.
In yet another embodiment, the reagent is a compound of formula 3:
Wherein n is 1 or 2; m is 1 or 2; A is R2COxe2x80x94, R3xe2x80x94Oxe2x80x94COxe2x80x94, or R4SO2xe2x80x94; a group of the formula: 
further wherein R1 is a hydrogen atom, alkyl or phenylalkyl; R2 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R3 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R5 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R6 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R7 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R8 is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula:
xe2x80x94CH2XR9; 
wherein R9 is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94(ARYL); 
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94(HETEROARYL); 
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94PO(R10)R11 
wherein R10 and R11 are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
In another aspect, the present invention provides a composition comprising a cosmetic reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family and a cosmetically or dermatologically acceptable carrier, adapted for preventing or ameliorating irritation of the skin of a mammal due to said cosmetic. In certain embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In other embodiments, the reagent is a compound of formula 1 as disclosed above. In yet other embodiments, the reagent is a compound of formula 3 as disclosed above.
In another aspect, the present invention provides a method for preventing or ameliorating inflammation due to contact of the skin of a mammal with an irritant by contacting the skin with a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family. In one embodiment, the irritant is a chemical irritant. In further embodiments, the chemical irritant is a cosmetic one or one from a plant. In yet further embodiments, the plant is selected from the group consisting of Poison Ivy, Poison Oak, and Poison Sumac. In another embodiment, the irritant is radiation. In one further embodiment, the radiation is ultraviolet radiation. In yet other embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In still other embodiments, the reagent is a compound represented by formula 1 or formula 3 as discussed above.
In another aspect, the present invention provides a composition that comprises a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family formulated for topical administration for use in preventing or ameliorating inflammation due to skin irritation. In one embodiment, the formulation is selected from a lotion, a cream, a gel, a liquid, a solid, or a semisolid. In another embodiment, the skin irritation is due to contact of the skin with a chemical irritant. In a further embodiment, the chemical irritant is a cosmetic or an agent derived from a plant. In yet another embodiment, the irritant is radiation. In certain embodiments, the irritation is due to an insect sting, an insect bite or tissue damage. In further embodiments, the tissue damage is due to physical trauma or disease. In yet further embodiments, the tissue (Physical trauma or disease) damage is selected from the group consisting of a burn, a scrape, a cut, frostbite, and chemical injury. In certain embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In certain embodiments, the reagent is a compound represented by formula 1 or formula 3 as discussed above.
In another aspect, the present invention provides a method for preventing or ameliorating inflammation due to contact of a tissue of a mammal with an irritant by contacting said tissue with a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family. In one embodiment, the irritant is a chemical irritant. In further embodiments, the chemical irritant is a cosmetic or an agent from a plant. In yet further embodiments, the plant is selected from the group consisting of Poison Ivy, Poison Oak, and Poison Sumac. In another embodiment, the irritant is radiation. In further embodiment, the radiation is ultraviolet radiation. In yet another embodiment, the irritant is a bacterium. In other embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In certain embodiments, the reagent is a compound represented by formula 1 or formula 3 as discussed above.
In another aspect, the present invention provides a method for preventing or ameliorating inflammation associated with tissue damage by contacting said tissue with a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family. In certain embodiments, the tissue damage is due to physical trauma, an autoimmune response, an infectious disease, chronic disease, spinal or brain trauma, an acid, a base, or radiation. In other embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In yet other embodiments, the reagent is a compound represented by formula 1 or formula 3 as discussed above.
In another aspect, the present invention provides a composition that comprises a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family and a pharmaceutical, dermatological, or cosmetic carrier formulated for topical application to the skin or mucus membrane of an animal. In one embodiment, the composition ameliorates symptoms associated with an inflammatory response. In a further embodiment, the symptoms comprise itching, redness, or swelling. In another embodiment, the composition is useful in decreasing loss of collagen or maintaining skin elasticity and appearance. In certain embodiments, the reagent suppresses the protease activity in an irreversible manner or reversible manner. In yet certain embodiments, the reagent is a compound represented by formula 1 or formula 3 as discussed above.
In yet another aspect, the present invention provides a method for reducing inflammation of a tissue by contacting said tissue with an effective amount of a reagent that suppresses the protease activity of at least one member of the interleukin-1beta-converting enzyme (ICE)/CED-3 family, thereby reducing inflammation of said tissue. In one embodiment, the tissue is skin. In a further embodiment, the tissue inflammation is due to trauma, sunburn, eczema, contact allergy, dermatitis, psoriasis, erysipelas, acne, ingrown nails, cuts, burns, insect bites, insect stings, or pruritus. In another embodiment, the tissue is mucosa. In a further embodiment, the tissue inflammation is due to vaginitis, hemorrhoids, conjunctivitis, periodontitis, wisdom tooth eruption, teeth extraction, gingivitis, periodontal abscesses, or prosthesis.
In further aspects, the present invention provides methods for ameliorating and/or treating infectious disease or symptoms (e.g., inflammation) associated therewith by contacting a cell or cell populations at risk of or exposed to an infectious agent with an inhibiting or suppressive amount of a reagent that suppresses or diminishes the protease activity of at least one member of the interleukin-1xcex2-converting enzyme (ICE/CED-3) family.
In certain embodiments, the methods can be used in vitro or in vivo. In other embodiments, the infectious agent may be a virus, a fungus, or a bacteria. In yet other embodiments, the reagent suppresses the protease activity in an irreversible or reversible manner.
Exemplary compounds useful as ICE/CED-3 inhibitors are also included herein. Such compounds and methods of synthesis are described in their entirety in co-pending U.S. patent application Ser. Nos. 09/482,813 filed Jan. 13, 2000, 09/550,917 filed Apr. 17, 2000, 09/747,317, filed Dec. 20, 2000, 09/908,969 filed Jul. 18, 2001, U.S. Pat. No. 5,869,519 issued Feb. 9, 1999, U.S. Pat. No. 5,877,197 issued Mar. 2, 1999, U.S. Pat. No. 6,184,244 issued Feb. 6, 2001, U.S. Pat. No. 6,187,771 issued Feb. 13, 2001, U.S. Pat. No. 6,197,750 issued Mar. 6, 2001, U.S. Pat. No. 6,242,422 issued Jun. 5, 2001 and their respective continuations-in-part.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.